Vitamin C may reduce troponin and CKMB levels after PCI and CABG: a meta-analysis

Background Ischemia/reperfusion injury contributes to periprocedural myocardial injury (PMI) in patients undergoing percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG). PMI can be estimated by the elevation of troponin (Tn) and creatine kinase-MB (CKMB) plasma levels, and it is associated with increased risk of cardiovascular events and mortality. Vitamin C might have a beneficial effect on PMI by improving endothelial function, improving myocardial perfusion, and by reducing oxidative stress generated during/after reperfusion. In several small animal models of cardiac stress, vitamin C reduced the increase in Tn and CKMB levels. The aim of this meta-analysis was to investigate whether vitamin C administration may have an effect on Tn and CKMB levels in patients undergoing PCI or CABG. Methods We searched PubMed, Cochrane, Embase and Scopus databases for controlled clinical trials reporting on Tn and CKMB levels in adult patients who underwent PCI or CABG and received vitamin C. As secondary outcomes we collected data on biomarkers of oxidative stress in the included trials. In our meta-analysis, we used the relative scale and estimated the effect as the ratio of means. Results We found seven controlled trials which included 872 patients. All included trials administered vitamin C intravenously, with a range from 1 to 16 g/day, and all initiated vitamin administration prior to the procedure. Vitamin C decreased peak Tn plasma levels in four trials on average by 43% (95% CI: 13 to 63%, p = 0.01) and peak CKMB plasma levels in five trials by 14% (95% CI: 8 to 21%, p < 0.001). Vitamin C also significantly decreased the biomarkers of oxidative stress. Conclusions Vitamin C may decrease cardiac enzyme levels in patients undergoing elective PCI or CABG. This may be explained partially by its antioxidant effects. Our findings encourage further research on vitamin C administration during cardiac procedures and in other clinical contexts that increase the level of cardiac enzymes. Future studies should search for an optimal dosing regimen, taking baseline and follow-up plasma vitamin C levels into account. Supplementary Information The online version contains supplementary material available at 10.1186/s12872-023-03459-6.


Calculations with data of the included trials
In Additional file 2, all calculations can be found of the included trials.In the Notes section of each trials above, the motivation for the data used is decribed.Here, we give explanatory notes on the different worksheets and formulas used in Excel.

Worksheet 1 -Primary endpoint
Worksheet 1 summarizes all data used for our primary endpoint.To calculate RoM, we need means ± SD for each group in each trial.For two trials, Wang and Basili, only skewed data was reported.Therefore, we estimated the mean for each group by adding the first quartile, the mean and the third quartile, and then dividing by 3 (S1).Now the RoM was calculated: the ratio of the means.
For the meta-analysis, the natural logaritm of RoM (treatment effect (TE)) and its standard error (SE) were calculated, according to the literature (S2).To check the correctness of the formula, an example of Friedrich was taken (shown in blue line).When SD was not available, due to skewed data in Wang and Basili, the SE of the TE was estimated by using the reported p-value.In the tabel, the Z-score of these p-values is shown.With the Z-score the SE can be estimated (TE/Z-score = SE).This is shown in the table below.Now all the SE's are estimated, the 95% confidence interval of the RoM can be calculated, as shown in the last two collumns.
Oktar group III was also added, as these data was used in the sensitivity analysis.

Worksheet 2 -Dingchao curve measurement
Dingchao only reported data in a curve.As described above, the nummerical data could be retrieved from the curve with the use of the GIMP graphics program.As a reference, the pixels of 90 and 10 IU/L were estimated first, after which the IU/L of the peaks CKMB could be estimated based on the measured pixels.This was also done for the CKMB levels at 90 and 120 minutes, as these data will be used on worksheet 3.

Worksheet 3 -Oktar_Dingchao SD
We suspected that the dispersion parameter in the curves of Oktar and Dingchao is SE, not SD as mentioned in the paper.
In Oktar, we recalculated the p-value of the difference in CKMB at 48h (group II-IV) while assuming SD is SE (scenario 1) and SD is SD (scenario 2).The p-value of scenario 2 was very small (1,04243E-09), and the p-value of scenario 1 (p=0.08)was close to the reported p-value of 0.03.This was also concluded for the p-value of the difference at 36h (group II-IV) when assuming SD is SE.Therefore, the reported dispersion of the CKMB results were considered to be SE's.Thus, SE was used to calculate the SD for all time points as shown in Table 2 in this worksheet.The yellow marked data was then used in worksheet 1.
In Dingchao, we recalculated the p-value of the difference in CKMB levels at 90 minutes (nonsignificant in the paper) while assuming that the reported SD is SE (scenario 1), and while assuming that SD is SD (scenario 2).The data from worksheet 2 was used.The p-value of scenario 2 was very small, and the p-value of scenario 1 was close to what is expected from the curve.This was also concluded for the p-value of the difference at 120 min when assuming SD is SE.Therefore, the peak CKMB levels and SE was measured from the curve.SE was then used to calculate the SD, which was used in worksheet 1.

Worksheet 4 -Secondary endpoint
For 8-Ohdg and 8-iso-PGF2alpha the RoM with 95% confidence interval was calculated in exactly the same way as described for worksheet 1.  1 and 2).

Allocation concealment (selection bias)
Low risk See above.
Blinding of participants and personnel (performance bias) Low risk Not described.It seems highly unlikely that knowledge of treatment might influence the levels of the measured biomarkers.Dr. Antonic confirmed there was no blinding (mail 2016-12-01).

Blinding of outcome assessment (detection bias)
Low risk Dr. Antonic confirmed there was no blinding (mail 2016-12-01).However, since our outcome is a laboratory measure, we judged that the possibility of performance and detection bias was low.

Incomplete outcome data (attrition bias)
Low risk No description of drop-outs.The short trial duration and the fact that the 'hospital length of stay' was measured for all included patients suggest no drop-outs.No drop-outs comfirmed by Dr. Antonic (mail 2016-12-01) Selective reporting (reporting bias)

Low risk
The study protocol is not available.However, Tn is currently the most essential assay for cardiac damage and it does not seem reasonable to assume that Tn is selected from a large set of cardiac biomarkers, in particular as the result is negative.The effect of intravenous vitamin C in patients with ischemia/reperfusion injury 27-Aug-2023

Timing of cardiac enzyme measurement
Baseline and every 6 h over 2 days after PCI, and thereafter if these were abnormal values.

Notes
Only the median absolute increase in TnI from baseline was reported.We were able to contact Prof Violi for the baseline and 6 h troponin data, but data was not available any more (email 2020-11-3).We assumed that the baseline TnI levels in the vitamin C and control patients were balanced, and therefore the increase from baseline is a valid measure of the difference between the two groups.

TnI:
Troponin The reported lower quartile level was 0.02 and 0.05 but that has to be an error since lower IQR limit cannot be greater than median.We assumed a typographical error so that one zero had been dropped.We used the reported p-value of 0.0832 to calculate Z = 1.73, from which we estimated the SE(TE).
See the Excel file for the calculations and page 3 of this document.

Risk of bias table
Bias Authors' judgement Support for judgement

Allocation concealment (selection bias)
Low risk "opaque envelopes containing a computer-generated random sequence were used for randomization" (Pignatelli 2011, p387).See also below.

Blinding of participants and personnel (performance bias)
Low risk "The operators who performed the (angiographic) evaluation were unaware of the study protocol and of the patient's characteristics'' (Basili 2010, p224)."Laboratory analysis: To ensure blind analysis, cardiologists sent tube identified by numerical code to the laboratory where biologists perform analytical tests.The randomization list was unveiled after that the analytical phase was terminated'' The effect of intravenous vitamin C in patients with ischemia/reperfusion injury 27-Aug-2023 Review Manager 5.3 4 (Pignatelli 2011, p387;also mentioned in Basili 2011.

Blinding of outcome assessment (detection bias)
Low risk See above.
Selective reporting (reporting bias) Low risk ''Serum cardiac-Troponin I (cTpI) was measured at baseline before the procedure, every 6 h over the next 2 days, and thereafter if these were abnormal values'' (Basili 2010, p223

Exclusion:
Recent MI, angina resistant to medical treatment,Ca channel blockers, medication with antioxidant properties, LVEF < 40%, additional organ dysfunction.

Interventions
Vit C: 0.05 g/kg of intravenous vitamin C just after the induction of anaesthesia and just before aortic declamping.Thus, the total dose was 0.1 g/kg.Assuming a 65 kg patient, this corresponds to 6.5 g/day on a single day.

Control:
Not described (Group C).

Timing of cardiac enzyme measurement
Baseline and 2h after surgery

Notes
Table 2: CKMB: VitC: 68.6 SD 16.3; Control: 74.1 SD 21.0 There was also a third group of patients who received vitamin C together with diltiazem.This group was excluded from our analysis.

Risk of bias table
Bias Authors' judgement Support for judgement

Random sequence generation (selection bias)
Low risk 'The patients were randomly divided into three groups, 10 patients each'.(p69).The report published 10 + 10 + 10 which indicates that all randomized participants were included.The baseline age, LVEF, grafts/patient were balanced (Table 1).

Allocation concealment (selection bias) Unclear risk Not described
Blinding of participants and personnel (performance bias) Low risk Not described.It seems highly unlikely that knowledge of treatment might influence the levels of the measured cardiac enzymes.

Blinding of outcome assessment (detection bias)
Low risk See above.

Incomplete outcome data (attrition bias)
Unclear risk No flowchart, no description of any dropouts.

Low risk
The study protocol is not available The goal of the authors was to compare the protective effects ...on ischaemia-reperfusion injury (p68).CKMB was a standard test for evaluating possible cardiac damage at that time and was published.It seems implausible that CKMB was selected from a large set of cardiac markers.

Notes
We measured the peak CKMB levels and their dispersion from Figure 1 by using the GIMP graphics program.However, we concluded that the dispersion parameter in the curves is SE, not SD as mentioned in the paper (last sentence method section).We recalculated the p-value of the difference in CKMB levels at 90 minutes (non-significant in the paper) while assuming that the reported SD is SE (scenario 1), and while assuming that SD is SD (scenario 2), see Additional File 2. The p-value of scenario 2 was very small, and the p-value of scenario 1 was close to what is expected from the curve.This was also concluded for the p-value of the difference at 120 min when assuming SD is SE.
Therefore, the peak CKMB levels and SE were measured from Figure 1.SE was then used to calculate the SD.See Additional File 2.

CKMB:
VitC In our manuscript we also compared groups II and III against the control group IV.Furthermore, in a sensitivity analysis we carried out a meta-analysis of the effect of vitamin C on CKMB using the vitamin C group III.Results did not differ considerably, see printouts of statistical calculations at page 5 of this document.
Oktar ( 2001) study also included a fourth group I, which was administered vitamin E, which is not included in our analysis.
CKMB levels were extracted from Table 2.We concluded that the dispersion of CKMB Table 2 was SE and not SD, though SD was reported.In Additional File 2 we recalculated the p-values of the differences in CKMB at 48h (group II-IV) while assuming SD is SE (scenario 1) and SD is SD (scenario 2).The p-value of scenario 2 was very small (1,0E-09), whereas the p-value of scenario 1 (p=0.08)was close to the reported p-value of 0.03.This was also concluded for the

on CKMB: sensitivity analysis using vitamin C group III of Oktar (2001)
Details on meta-analytical method:-Inverse variance method -Restricted maximum-likelihood estimator for tau^2 -Q-profile method for confidence interval of tau^2 and tau Review Manager 5.3 1 ''We used random.orgonline service'' and patients were included in a strict order with no exception.Dr. Antonic provided this information (mail 2016-12-01).The groups were well matched for age, sex, EuroSCORE II (European System for Cardiac Opeative Risk Evaluation), DM2, serum creatinine, preoperative beta-blocker and statin, and the number of bypass grafts.(Tables ).However, Basili does not describe what were the time points for which they reported the troponin values.The TnI values were published even though the result was negative.
Intravenously 4 g of vitamin C just before the induction of anaesthesia (Group II) Control: Not described, suggesting no saline as a placebo (Group IV).(in Group III 4 g of vitamin C was added to the cardioplegic solution)NotesIn our study, we compared group II with group IV.There is another vitamin C group (group III) who were administered 4 g of vitamin C in the cardioplegic solution.We used group II because vitamin C was administered earlier compared to group III, and earlier administration can lead to more effective body distribution.